V-ATPases are conserved proton pumps important to pH homeostasis. Located at the membrane of lysosomes, vacuoles and endosomes, V-ATPases sustain the acidic luminal pH needed for protein sorting and degradation; and for entry of viruses and bacterial toxins into host cells. Cells specialized for active proton secretion such as the 1-intercalated cells of the kidney nephron, express V-ATPases at the plasma membrane where they fine-tune the systemic acid-base balance. It is our goal to demonstrate the fundamental mechanisms that carefully regulate V-ATPase function and assembly in order to gain insight into how V- ATPases assist in controlling luminal, cytosolic, and extracellular pH. V-ATPases are dynamic structures that reversibly disassemble to control pH. In yeast and kidney cells, activity and V-ATPase assembly are coupled with glycolysis, but the mechanisms involved are unclear. Using yeast model systems, we have shown a novel link between V-ATPases and the glucose-fatty acid cycle, suggesting that glucose and lipid metabolism remodel pH homeostasis via effects on V-ATPases. It is our hypothesis that V-ATPase assembly is regulated as a means of maintaining cellular pH homeostasis when metabolism switches between glucose and fatty acids. We propose that a complex consisting of the V-ATPase pump and glycolytic enzymes functionally and structurally couples pH homeostasis and energy metabolism; and that metabolic control of this macromolecular structure regulates V-ATPase assembly. Three specific aims will test this model: Aim 1 will dissect the metabolic signals that link V-ATPase to the glycolytic pathway; Aim 2 will elucidate the mechanisms by which regulation of glycolytic enzymes remodels V-ATPase assembly and activity; and Aim 3 will establish how activation of the glucose-fatty acid cycle cross-talks to V-ATPases. In order to accomplish the aims proposed, this study will measure V-ATPase assembly and disassembly in vma mutants and metabolic mutants deficient in key steps of glycolysis, 2- oxidation of fatty acids, and the glucose-fatty acid cycle. Parallels will be established between intracellular levels of metabolic intermediates and dynamics of binding between V-ATPase and glycolytic enzymes enabling us to understand how V-ATPases assist cells in adjusting to metabolic changes. Because of the complexity involved in both comprehensive metabolic studies and regulation of V-ATPase pumps by reversible disassembly, S. cerevisiae is an outstanding system to address this mechanism at both the genetic and biochemical levels. By showing the contribution of V-ATPases, new insights into the mechanisms that tune fuel energy selection will emerge. Cancer cells use V-ATPases to regulate pH as a result of changes in metabolism; thus new knowledge on the mechanisms by which V-ATPases maintain pH homeostasis in cancer may be revealed. As pathophysiology of the glucose-fatty acid cycle results in metabolic disorders including diabetes and chronic kidney disease, our studies will also contribute towards their understanding.